Injectable Cross-Linked Polymeric Preparations and Uses Thereof

ABSTRACT

A therapeutic composition for treatment of a body tissue which includes an aqueous solution of a cross-linked polymer being capable of: (i) maintaining a liquid state in storage at room temperature for at least 24 hours; and (ii) assuming a gel state following deposition within the body tissue. The therapeutic composition can be effectively administered into a damaged body tissue via injection or catheterization, thereby treating the damaged body tissue.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/430,020, filed on Mar. 26, 2012, which is a Continuation-In-Part ofU.S. patent application Ser. No. 11/229,119, filed on Sep. 19, 2005, nowU.S. Pat. No. 8,168,612, which is a Continuation-In-Part of U.S. patentapplication Ser. No. 10/840,008, filed on May 5, 2004, which claimspriority of Israel Patent Application No. 155774, filed on May 5, 2003.U.S. patent application Ser. No. 11/229,119 is also aContinuation-In-Part of PCT Patent Application No. PCT/IL2004/000371,filed on May 4, 2004. The contents of the above Applications are allincorporated by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to cross-linked polymer compositionscapable of treating body tissues and, more particularly, to aqueouscross-linked polymer solutions and methods of use thereof.

Cross-linked polymer gel materials are widely utilized in the biomedicalindustry. For example, polysaccharide gels have been applied in contactlenses, blood contact materials, controlled release formulations, wounddressings, bioadhesives, membranes, superabsorbents, cell encapsulationand immunoisolation materials, and tissue engineering scaffolds (Suggset al., J. Biomater. Sci. Polym. 9: 653-666, 1998; Aebischer et al.,Transplantation 58: 1275-1277, 1994; and Atala et al. (J. Urol. 150:745-747, 1993).

The potential use of polysaccharide gel materials for treating damagedheart tissue has been intensively researched during the past decade.

The main focus of research has been on utilizing polysaccharide gels fortreating the heart tissue following myocardial infarction (MI). MItypically causes an acute loss of myocardial tissue and an abruptincrease in loading conditions which induces left ventricular (LV)remodeling. The early phase of LV remodeling involves expansion of theinfarct zone, which often results in early ventricular rupture oraneurysm formation. Late remodeling encompasses the entire LV andinvolves time-dependent dilatation, recruitment of border zonemyocardium into the scar, distortion of ventricular shape and muralhypertrophy. Consequently, it may lead to progressive deterioration incontractile function, heart failure and eventually death (Sutton andSharpe, Circulation 101:2981-2988, 2000; Mann, D. L., Circulation100:999-1008, 1999; and Jugdutt, B. I., Circulation 108:1395-1403,2003).

Accordingly, cessation or reversal of progressive chamber remodeling isan important aim of heart failure therapy. Clinical attempts to minimizethe devastating effects of MI have thus far failed to effectively repairthe irreversible damage inflicted to the heart tissue (Khand et al.,Eur. Heart J. 22:153-164, 2001; Jessup and Brozena, S. N. Engl. J. Med.348:2007-2018, 2003; and Redfield, M. M., N. Engl. J. Med. 347:1442-1444, 2000).

Recently, attempts to implant living cells in damaged myocardium havegiven hope for repairing the damaged tissue via promoting tissueregeneration (Etzion et al., J. Mol. Cell Cardiol. 33:1321-1330, 2000;Leor et al., Expert Opin. Biol. Ther. 3:1023-39, 2003; and Beltrami etal., Cell; 114:763-776, 2003). This approach has advanced considerablywith the development of 3-D biomaterial scaffolds aimed at supportingimplantation of donor cells (e.g., cardiac cells or stem cells) in themyocardium. Lately, 3-D biomaterial scaffolds made of polysaccharide gelwere successfully implanted onto damaged myocardium with promisingresults (Leor et al., Circulation 102:56-61, 2000). However, clinicaluse of such cell seeded 3-D biomaterial scaffolds is limited due toscarcity of suitable donor cells and the high risk involved in majorsurgery.

While reducing the present invention to practice, the present inventorsgenerated a stable solution of a cross-linked polymer which can besafely administered into a body tissue, such as a damaged myocardium,using low invasive techniques and which can effectively repair thedamaged tissue.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided atherapeutic composition for treatment of a body tissue. The therapeuticcomposition includes an aqueous solution of a cross-linked polymer beingcapable of: (i) maintaining a liquid state in storage at roomtemperature for at least 24 hours; and (ii) assuming a gel statefollowing deposition within the body tissue.

According to another aspect of the present invention there is provided atherapeutic composition for treatment of a body tissue. The therapeuticcomposition includes an aqueous solution of a cross-linked polymer beingcapable of: (i) maintaining a liquid state within a blood vessel; and(ii) assuming a gel state following deposition within the body tissue.

According to yet another aspect of the present invention there isprovided an article of manufacturing, including the therapeuticcomposition and a packaging material identifying the therapeuticcomposition for use in tissue repair.

According to still another aspect of the present invention there isprovided a kit for treatment of a body tissue. The kit includes: (a) thetherapeutic composition; (b) a device suitable for administering thetherapeutic composition into the body tissue; and (c) a packagingmaterial identifying the kit for use in treatment of the body tissue.

According to an additional aspect of the present invention there isprovided a method of producing a therapeutic composition for treatmentof a body tissue. The method is effected by: (a) providing an aqueoussolution containing a predetermined multivalent cation salt to polymersalt ratio; and (b) mixing the aqueous solution under conditionssuitable for uniformly cross linking the polymer of the polymer saltwith the multivalent cation of the multivalent cation salt and yetmaintains the aqueous solution as an aqueous cross-linked polymersolution, thereby producing the therapeutic composition for treatment ofa body tissue.

According to yet an additional aspect of the present invention there isprovided a method of treating a damaged body tissue. The method iseffected by providing the damaged body tissue with an effective amountof the therapeutic composition.

According to still an additional aspect of the present invention thereis provided a method of treating a heart condition. The method iseffected by providing a heart tissue with an effective amount of thetherapeutic composition.

According to a further aspect of the present invention there is provideda method of inducing angiogenesis in a damaged heart tissue. The methodis effected by providing the damaged heart tissue with an effectiveamount of the therapeutic composition.

According to further features in preferred embodiments of the inventiondescribed below, the therapeutic composition is further capable ofmaintaining a liquid state in storage at a temperature ranging fromabout 4 to about 8° c. for at least 30 days.

According to still further features in the described preferredembodiments the therapeutic composition is further being capable ofmaintaining the liquid state within a blood vessel.

According to still further features in the described preferredembodiments the therapeutic composition is further being capable ofspreading from the blood vessel to the body tissue.

According to still further features in the described preferredembodiments the aqueous solution of a cross-linked polymer isadministratable into the body tissue via a needle.

According to still further features in the described preferredembodiments the needle has an 18-27 gauge bore.

According to still further features in the described preferredembodiments the aqueous solution of a cross-linked polymer isadministratable via infusion or catheterization.

According to still further features in the described preferredembodiments the blood vessel is an artery.

According to still further features in the described preferredembodiments the artery is a coronary artery.

According to still further features in the described preferredembodiments the aqueous solution of a cross-linked polymer exhibits: (i)an elastic response being equal to or greater than its viscous responseunder small deformation oscillatory frequencies in the linearviscoelastic limit; and (ii) shear thinning behavior in a powerlawrelationship.

According to still further features in the described preferredembodiments the small deformation oscillatory frequencies range from0.01 to 100 Hz.

According to still further features in the described preferredembodiments the small deformation oscillatory frequencies range from 0.1to 10 Hz.

According to still further features in the described preferredembodiments the polymer is a polysaccharide.

According to still further features in the described preferredembodiments the polysaccharide is an alginate.

According to still further features in the described preferredembodiments the alginate has a molecular weight ranging from 1 to 300kDa.

According to still further features in the described preferredembodiments the alginate has a molecular weight ranging from 5 to 200kDa.

According to still further features in the described preferredembodiments the alginate has a molecular weight ranging from 10 to 100kDa.

According to still further features in the described preferredembodiments the alginate has a molecular weight ranging from 20 to 50kDa.

According to still further features in the described preferredembodiments a concentration of the alginate in the aqueous solution of across-linked polymer ranges from 0.1 to 4% (w/v).

According to still further features in the described preferredembodiments a concentration of the alginate in the aqueous solution of across-linked polymer ranges from 0.5 to 2% (w/v).

According to still further features in the described preferredembodiments a concentration of the alginate in the aqueous solution of across-linked polymer ranges from 0.8 to 1.5% (w/v).

According to still further features in the described preferredembodiments a concentration of the alginate in the aqueous solution of across-linked polymer is about 1% (w/v).

According to still further features in the described preferredembodiments the polymer is cross-linked by multivalent cations.

According to still further features in the described preferredembodiments the multivalent cations are uniformly distributed within thepolymer.

According to still further features in the described preferredembodiments the multivalent cations are calcium cations.

According to still further features in the described preferredembodiments a concentration of the calcium cations in the aqueoussolution of a cross-linked polymer ranges from 0.005 to 0.1% (w/v).

According to still further features in the described preferredembodiments a concentration of the calcium cations in the aqueoussolution of a cross-linked polymer ranges from 0.01 to 0.05% (w/v).

According to still further features in the described preferredembodiments a concentration of the calcium cations in the aqueoussolution of a cross-linked polymer ranges from 0.02 to 0.04% (w/v).

According to still further features in the described preferredembodiments a concentration of the calcium cations in the aqueoussolution of a cross-linked polymer ranges from 0.025 to 0.035% (w/v).

According to still further features in the described preferredembodiments a monomer ratio between α-L-guluronic acid andβ-D-mannuronic acid in the alginate ranges between 1:1 and 3:1.

According to still further features in the described preferredembodiments a monomer ratio between α-L-guluronic acid andβ-D-mannuronic acid in the alginate ranges between 1.5:1 and 2.5:1.

According to still further features in the described preferredembodiments a monomer ratio between α-L-guluronic acid andβ-D-mannuronic acid in the alginate is about 2.

According to still further features in the described preferredembodiments the therapeutic composition further includes cells.

According to still further features in the described preferredembodiments the, cells are selected from the group consisting ofcardiomyocetes, myoblasts, fibroblasts, chondrocytes, muscle cells,smooth muscle cells, endothelial cells, mesenchymal cells and stemcells.

According to still further features in the described preferredembodiments the therapeutic composition further includes at least onetherapeutic agent.

According to still further features in the described preferredembodiments the at least one therapeutic agent is selected from thegroup consisting of a growth factor, a hormone, an anti-inflammatorydrug, an anti-apoptotic drug and an antibiotic drug.

According to still further features in the described preferredembodiments the tissue is a myocardial tissue.

According to still further features in the described preferredembodiments the tissue is muscle tissue.

According to still further features in the described preferredembodiments the body tissue is a myocardial body tissue.

According to still further features in the described preferredembodiments the body tissue is a muscle tissue.

According to still further features in the described preferredembodiments the device includes a syringe.

According to still further features in the described preferredembodiments the syringe is equipped with an 18-27 gauge bore needle.

According to still further features in the described preferredembodiments the device includes a catheter.

According to still further features in the described preferredembodiments the catheter is suitable for intra-arterial administrationof the therapeutic composition.

According to still further features in the described preferredembodiments the catheter is suitable for intra-coronary administrationof the therapeutic composition.

According to still further features in the described preferredembodiments the catheter suitable for intra-coronary administration isan over-the-wire balloon catheter.

According to still further features in the described preferredembodiments the polymer salt is a polysaccharide salt.

According to still further features in the described preferredembodiments the polysaccharide salt is an alginate salt.

According to still further features in the described preferredembodiments the polymer salt is sodium alginate.

According to still further features in the described preferredembodiments the multivalent cation salt is a calcium salt.

According to still further features in the described preferredembodiments the calcium salt is calcium gluconate.

According to still further features in the described preferredembodiments the predetermined multivalent cation salt to polymer saltratio ranges between 2:1 and 1:10.

According to still further features in the described preferredembodiments the predetermined multivalent cation salt to polymer saltratio ranges between 1:1 and 1:6.

According to still further features in the described preferredembodiments the predetermined multivalent cation salt to polymer saltratio ranges between 1:2 and 1:5.

According to still further features in the described preferredembodiments the predetermined multivalent cation salt to polymer saltratio ranges between 1:3 and 1:4.

According to still further features in the described preferredembodiments the mixing is effected by using a homogenizer.

According to still further features in the described preferredembodiments the homogenizer is operated at a speed setting ranging from2,000 to 50,000 rpm.

According to still further features in the described preferredembodiments the homogenizer is operated at a speed setting ranging from5,000 to 40,000 rpm.

According to still further features in the described preferredembodiments the mixing is effected at a speed setting ranging from10,000 to 30;000 rpm.

According to still further features in the described preferredembodiments the mixing is effected over a period of at least 30 seconds.

According to still further features in the described preferredembodiments the mixing is effected over a period of at least 1 minute.

According to still further features in the described preferredembodiments the mixing is effected over a period of at least 2 minutes.

According to still further features in the described preferredembodiments the damaged body tissue is a damaged myocardial tissue.

According to still further features in the described preferredembodiments the myocardial tissue is the left ventricular wall tissue.

According to still further features in the described preferredembodiments the damaged body tissue is a damaged muscle tissue.

According to still further features in the described preferredembodiments the step of providing is effected via a needle.

According to still further features in the described preferredembodiments the step of providing is effected intra-arterially via asuitable catheter.

According to still further features in the described preferredembodiments the step of providing is effected intra-coronarily via asuitable catheter.

According to still further features in the described preferredembodiments the suitable catheter is an over-the-wire balloon catheter.

According to still further features in the described preferredembodiments the effective amount ranges between about 0.1 and 10 ml.

According to still further features in the described preferredembodiments the effective amount ranges between about 0.5 and 5 ml.

According to still further features in the described preferredembodiments the effective amount ranges between 1 and 4 ml.

According to still further features in the described preferredembodiments the heart condition is a congestive heart failure or anischemic mitral regurgitation.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a therapeutically beneficialcross-linked polymer solution which can be administered into a damagedbody tissue via injection or catheterization, thereby providingsubstantial therapeutic benefits to the damaged body tissue safely andeffectively.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1A illustrates the effect of shear rate on viscosity of an aqueoussolution of 1% (w/v) sodium alginate (LF 5/60; viscosity=40 cP) mixedwith 0.3% (w/v) calcium 30 gluconate concentration. Abbreviations:visc., viscosity; S.R., Shear Rate;

FIG. 1B illustrates the effect of shear rate on viscosity of an aqueoussolution of 1% (w/v) sodium alginate (LF 5/60; viscosity=40 cP) mixedwith 0.4% (w/v) calcium gluconate concentration. Abbreviations: visc.,viscosity; S.R., Shear Rate;

FIG. 2 illustrates the effect of shear rate on viscosity of an aqueoussolution of 1% (w/v) sodium alginate (LVG; viscosity=127 cP) mixed with0.3% (w/v) calcium gluconate concentration. Abbreviations: visc.,viscosity; S.R., Shear Rate;

FIGS. 3A-C illustrate mechanical spectra of aqueous solutions of 1%alginate (LF 5/60; viscosity=40 cP) with no calcium gluconate added(FIG. 3A), mixed with 0.3% (w/v) calcium gluconate (FIG. 3B), or mixedwith 0.4% (w/v) calcium gluconate (FIG. 3C). Abbreviations: Freq.,frequency;

FIGS. 4A-B illustrate mechanical spectra of an aqueous solution of 1%(w/v) alginate (LVG; viscosity=127 cP) with no calcium gluconate added(FIG. 4A), or mixed with 0.3% (w/v) calcium gluconate (FIG. 4B).Abbreviations: Freq., frequency;

FIGS. 5A-H show echocardiography results illustrating the effect of theaqueous cross-linked alginate solution on the Left Ventricular (LV)remodeling. FIGS. 5A-B show the LV diastolic dimension M-mode in treatedand untreated animals, respectively. FIGS. 5C-D show the LV systolicdimension in treated and untreated animals, respectively. FIGS. 5E-Fshow the LV diastolic area in treated and untreated animals,respectively. FIGS. 5G-H show LV systolic area in treated and untreatedanimals, respectively. Abbreviations: B., baseline; 2 mo., two months;

FIGS. 6A-F show 2-D echocardiography results illustrating the effect ofthe aqueous cross-linked alginate solution on the Left Ventricular (LV)remodeling. FIGS. 6A-B show the AW 2-D in treated and untreated animals,respectively. FIGS. 6C-D show the LV diastolic dimension 2-D in treatedand untreated animals, respectively. FIGS. 6E-F show the LV systolicdimension 2-D in treated and untreated animals, respectively.Abbreviations: B., baseline; 2 mo., two months;

FIGS. 7A-D show echocardiography results illustrating the effect of theaqueous cross-linked alginate solution on the Left Ventricular (LV)function. FIGS. 7A-B show LV fractional shortening in treated anduntreated animals, respectively. FIGS. 7C-D show the LV fractional areachange in treated and untreated animals, respectively. Abbreviations:B., baseline; 2 mo., two months;

FIG. 8 illustrates the effect of the cross-linked alginate solution onneoangiogenesis (in number of vessels per area) in rat infractedmyocardium. Abbreviations: Ves., vessels; Alg., alginate; cont:,control;

FIGS. 9A-B illustrate the• effect of the cross-linked alginate solutionon reversing ischemic mitral regurgitation (MR). FIG. 9A is a schematicillustration of ischemic MR with mitral annulus dilatation. FIG. 9B is aschematic illustration of ischemic MR with change in the global geometryof the left ventricle and tethering of the mitral leaflet.

FIG. 10 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) with no calcium gluconate added.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 11 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.1% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 12 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.2% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 13 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.23% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 14 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.25% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 15 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.3% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 16 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.275% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 17 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.270% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 18 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=30 kDa) with no calcium gluconate added.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 19 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=100 kDa) mixed with 0.2% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 20 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=30 kDa) mixed with 0.3% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 21 illustrates a mechanical spectrum of an aqueous solution of 0.8%(w/v) alginate (Avg. Mw=30 kDa) with no calcium gluconate added.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 22 illustrates a mechanical spectrum of an aqueous solution of 0.8%(w/v) alginate (Avg. Mw=30 kDa) mixed with 0.2% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 23 illustrates a mechanical spectrum of an aqueous solution of 0.8%(w/v) alginate (Avg. Mw=30 kDa) mixed with 0.3% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 24 illustrates a mechanical spectrum of an aqueous solution of 1.5%(w/v) alginate (Avg. Mw=30 kDa) with no calcium gluconate added.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 25 illustrates a mechanical spectrum of an aqueous solution of 1.5%(w/v) alginate (Avg. Mw=30 kDa) mixed with 0.2% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 26 illustrates a mechanical spectrum of an aqueous solution of 1.5%(w/v) alginate (Avg. Mw=30 kDa) mixed with 0.3% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 27 illustrates a mechanical spectrum of an aqueous solution of 1.5%(w/v) alginate (Avg. Mw=30 kDa) mixed with 0.4% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 28 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=160 kDa) with no calcium gluconate added.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 29 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=160 kDa) mixed with 0.2% (w/v) calciumgluconate;

FIG. 30 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=15 kDa) with no calcium gluconate added.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 31 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=15 kDa) mixed with 0.2% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 32 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) to alginate (Avg. Mw=15 kDa) mixed with 0.3% (w/v) calciumgluconate. Circles=elastic response (G′); triangles=viscous response(G″); squares=complex viscosity;

FIG. 33 illustrates a mechanical spectrum of an aqueous solution of 1%(w/v) alginate (Avg. Mw=15 kDa) mixed with 0.4% (w/v) calcium gluconate.Circles=elastic response (G′); triangles=viscous response (G″);squares=complex viscosity;

FIG. 34 illustrates the effect of injecting the cross-linked alginatesolution (alginate) or PBS (control) into a mouse ischemic hind limb, onthe blood flow rate in the damaged tissue. Relative blood flow rateswere measured prior to injection and 7 days following injection;

FIG. 35 illustrates the effect of injecting the cross-linked alginatesolution (alginate) or PBS (control) into a mouse ischemic hind limb onthe limb survival. In the control group 3 out of 8 animals developednecrosis and subsequently fell off, while none of the alginate-treatedlimbs were lost;

FIG. 36 illustrates the effect of the cross-linked alginate solutioninjected into a rat myocardium two months post MI, on the averagemyocardial scar thickness measured four months post MI. The average scarthickness in the alginate-treated animals was 27.9±2.7 mm, compared withjust 16.3±3.1 in the PBS-treated 5 (control) animals (p<0.05);

FIG. 37 illustrates the effect of the cross-linked alginate solution,injected into rat myocardium two months post MI, on the average vesseldensity in the myocardial scar tissue measured four months post MI. Theaverage vessel density in the alginate-treated animals was 106±5vessels/mm², compared with just 43±8 vessels/mm² in the PBS-treated(control) animals (p<0.0001);

FIG. 38 illustrates the effect of the cross-linked alginate solutioninjected into a rat myocardium two months post MI, on the LV fractionalshortening (FS) measured by 2D-echocardiography four months post MI. Thefractional shortening in the PBS-treated (control) rats declined by37±9% (relative to the pre MI level), 20 compared with just 12±6% in thealginate-treated rats (p<0.0001); and

FIG. 39 illustrates the effect of the cross-linked alginate solution,injected into rat myocardium two months post MI, on the cardiacdiastolic function measured by E/A wave ratio in Doppler echocardiogramfour months post MI. The cardiac diastolic function in the PBS-treated(control) rats declined (relative to the pre MI level) by 37±9%,compared with just 12±6% in the alginate-treated rats (p<0.0001).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a cross-linked polymer solution which can beused to treat a body tissue, such as a damaged myocardial tissue.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

International patent application WO 99/15211 teaches an injectable,partially hardened hydrogel. This composition is viscoussuspension/paste capable of holding its shape against gravity. Thereference does not describe or suggest a cross-linked polymer solutionwhich is flowable and capable of stably maintaining a liquid state instorage.

U.S. Pat. No. 6,136,334 teaches an aqueous mixture comprising an ionicpolysaccharide capable of being gelled in situ upon contact with acounter ion. The reference does not describe or suggest a solution whichincludes a cross-linked polymer.

U.S. Pat. No. 5,709,854 teaches an injectable suspension ofpolysaccharide hydrogel mixed with living cells. The reference does notdescribe or suggest a cross-linked polymer solution which is flowableand capable of stably maintaining a liquid state in storage.

U.S. Pat. No. 6,592,886 teaches a cross-linked alginate gel matrix, suchas beads, for use in encapsulating transplant materials. The referencedoes not describe or suggest a cross-linked polymer solution which isflowable and capable of stably maintaining a liquid state in storage.

While reducing the present invention to practice the. present inventorssurprisingly and unexpectedly uncovered that alginate polysaccharide Canbe uniformly cross-linked to form a stable aqueous solution which isalso substantially viscoelastic. As a stable solution, the novelcomposition can be readily administered into a body tissue via a needleor a catheter. Most surprisingly, administering the solution of thepresent invention into animals' hearts, following myocardium infarct,resulted in remarkable improvement of heart function and regeneration ofdamaged myocardium tissue (see Examples 1-6 and 8 in the Examplessection which follows).

Thus, according to one aspect of the present invention, there isprovided a therapeutic composition for treatment of a body tissue. Thecomposition includes an aqueous solution of a cross-linked polymer beingcapable of (i) maintaining a liquid state in storage at room temperaturefor at least 24 hours and (ii) assuming a gel state following depositionwithin the body tissue.

As used herein, the term “solution” refers to a liquid in which two ormore substances (e.g. solute and solvent) are mixed together anduniformly dispersed. The phrase “aqueous solution” refers to a liquid inwhich one or more substances (solutes) are uniformly dispersed in water(solvent).

The terms “liquid” and “flowable” are used interchangeably herein andrefer to the capacity of a substance to flow freely and assume the shapeof the space containing it.

As used herein, the phrase “cross-linked polymer” refers to a network ofpolymer units being inter-linked via covalent, hydrogen or ionicbonding.

A suitable polymer, according to the teaching of the present invention,can be any biocompatible, non-immunogenic and, preferably, bio-erodiblepolymer which can be cross-linked to form a hydrogel.

As used herein, the term “gel” refers to a semisolid colloidalsuspension of a solid in a liquid. The term “hydrogel” refers to a gelwhich contains water as the liquid.

Preferably, the polymer of the present invention is a polysaccharide,most preferably an alginate.

The term “alginate” refers to a polyanionic polysaccharide copolymerderived from sea algae (e.g., Laminaria hyperborea, L. digitata, Ecloniamaxima, Macrocystis pyrifera, Lessonia nigrescens, Ascophyllum• codosum,L. japonica, Durvillaea antarctica, and D. potatorum) and which includesβ-D-mannuronic (M) and α-L-guluronic acid (G) residues in varyingproportions.

An alginate suitable for use in the present invention has a monomerratio between α-L-guluronic acid and β-D-mannuronic preferably rangingbetween 1:1 to 3:1, more preferably between 1.5:1 and 2.5:1, mostpreferably about 2.

An alginate suitable for use in the present invention has a molecularweight ranging preferably between 1 to 300 kDa, more preferably between5 to 200 kDa, more preferably between 10 to 100 kDa, most preferablybetween 20 to 50 kDa.

Cross linking of the polymer of the present invention can be effectedvia multivalent cations such as, but not limited to, calcium, strontium,barium, magnesium or aluminum, as well as di-, tri- and tetra-functionalorganic cations. In addition, polyions can be used such as, for example,poly(amino acids), poly(ethyleneimine), poly(vinylamine),poly(allylamine) and cationic polysaccharides. Most preferably, thecross linking is effected via calcium cations.

The multivalent cation salt is preferably a pharmacologically acceptablecalcium salt such as, for example, calcium gluconate, calcium citrate,calcium acetate, calcium fluoride, calcium phosphate, calcium tartrate,calcium sulfate, calcium borate or calcium chloride. Most preferably,the multivalent cation salt is calcium D-gluconate (calcium gluconate).Pharmaceutical grade calcium gluconate salts are readily available fromseveral commercial manufacturers such as, for example, Sigma.

The polymer salt is preferably a pharmacologically acceptable alginatesalt such sodium, potassium, lithium, rubidium and cesium salts ofalginic acid, as well as the ammonium salt, and the soluble alginates ofan organic base such as mono-, di-, or tri-ethanolamine alginates,aniline alginates, and the like. Most preferably, the polymer salt issodium alginate. Pharmaceutical grade sodium alginate salts, whichcomply with all the quality and safety requirements of the European andUnited States of America (USA) pharmacological regulatory authorities,are readily available from several commercial manufacturers such as, forexample, Novamatrix FMC Biopolymers (Drammen, Norway).

An aqueous solution containing calcium gluconate and sodium alginate ata predetermined ratio can be prepared by combining a sodium alginatestock solution with a calcium gluconate stock solution.

The weight ratio between calcium gluconate and sodium alginate in theaqueous solution preferably ranges between 2:1 and 1:10, more preferablybetween 1:1 and 1:6, more preferably between 1:2 and 1:5, mostpreferably between 1:3 and 1:4.

The aqueous cross-linked polymer solution of the invention can beobtained by uniformly cross linking the alginate particles via thecalcium cations being present in the aqueous solution. The cross linkingcan be effected by (i) providing an aqueous solution containing apredetermined multivalent cation salt to polymer salt ratio and (ii)mixing the aqueous solution under conditions suitable for uniformlycross linking the polymer with the multivalent cation and yetmaintaining the aqueous solution as an aqueous cross-linked polymersolution.

The phrase “uniformly cross linking” used herein refers to spreading thebonds linking the polymer chains in a substantially non-clustereddistribution, preferably random distribution, most preferably evendistribution. The uniformly crossed-linked solution assumes substantialviscoelasticity yet retains its liquidity and flowability.

Uniform cross linking can be effected by using a device (e.g.homogenizer) 10 capable of rigorously mixing the solution withoutsubstantially shearing the cross-linked polymer. A suitable homogenizercan be, for example, Heildolph DIAX 900 equipped with 10G dispenserhead. The homogenizer is operated at a working speed preferably rangingbetween 5,000 and 50,000 rpm, more preferably between 10,000 and 30,000rpm. Homogenization is conducted preferably for at least 30 seconds,more preferably at least 2 minutes, most preferably at least 5 minutes.

The therapeutic composition is can be prepared using the followingprocedure (see Example 1 hereinbelow for further details): (i) apredetermined amount of sodium alginate is mixed with a predeterminedvolume of distilled water; (ii) the mixture resulting from step (i) isstirred until a clear solution is obtained; (iii) the solution resultingfrom step (ii) is filter sterilized (e.g., using a 0.2 μm poremembrane); (iv) a predetermined amount of calcium D-Gluconate(hemicalcium salt) is mixed with a predetermined volume of distilledwater; (v) the mixture resulting from step (iv) is stirred until a clearsolution is obtained; (vi) the solution resulting from step (v) isfilter sterilized; (vii) the solution of step (iii) is combined thesolution of step (vi) and mixed using a suitable homogenizer to make thetherapeutic composition. Once prepared, the composition is stored at4-8° C. until use.

The final concentration (w/v) of alginate in the composition preferablyranges between 0.1 to 4%, more preferably between 0.5 and 2%, mostpreferably between 0.8 and 1.5%.

The final concentration (w/v) of calcium cations in the composition ofthe present invention preferably ranges between 0.005 and 0.1%, morepreferably between 0.01 and 0.05%, more preferably between 0.02 and0.04%, most preferably between 0.025 and 0.035%.

The therapeutic composition of the present invention preferably exhibitsan elastic response which is equal to or greater than its viscousresponse under small deformation oscillatory frequencies in the linearviscoelastic limit and a shear thinning behavior in a power-lawrelationship.

The term “viscosity (η)” used herein refers to a measure of theresistance of a fluid to flow. It is defined as the ratio of shearstress (τ) to shear rate (γ):

η=τ/γ

When the fluid obeys the equation for all shear rates, it is denotedNewtonian to [Ferry, J. D. (Ed.), Viscoelastic Properties of Polymers”,John Wiley & Sons, 1980].

The viscoelastic properties of the composition can be determined byapplying a sinusoidal stress or strain of frequency f to the sample andmeasuring the response.

The response is divided into (i) an elastic part in phase with theapplied stress or strain, and (ii) a viscous part out of phase. Becauseof the two components, a complex notation is used. The complex shearmodulus is denoted by G*, which is defined by the following formula:

G*=G′+jG″

wherein G′ is the storage modulus, i.e., the elastic part, G″ is theloss modulus (the viscous part), and j²=−1.

The shear modulus as a function of frequency can be expressed by theslope n in a log-log plot of G′ versus frequency, f, denoted by thefollowing formula:

Log G′=n log f+K

wherein K is a constant. In a physical gel n>0, in a covalent gel n=0.

Viscoelastic features can be presented in terms of the storage modulusG′ (herein referred to as the “elastic response”) and the loss modulusG″ (herein referred to as the “viscous response”) as a function ofangular frequency.

The values of elastic response (G′), viscous response (G′) and viscosity(ii) can be determined using standard rheological methods [see, forexample, Ronald G Larson, “The Structure and Rheology of ComplexFluids”, Oxford University Press, Inc., 663 pp., 1998; and ChristopherW. Macosko, “Rheology: Principles, Measurements, and Applications”,Wiley-VCH Inc., 568 pp., 1994].

Rheological measurements are preferably obtained under an oscillatoryfrequency ranging within the viscoelastic limit, preferably rangingbetween 0.01 and 100 Hz, more preferably ranging between 0.1 to 10 Hz.

As illustrated in Examples 1 hereinbelow, preferred compositions of thepresent invention do not have permanent cross links, are stronglyfrequency dependent and have G′-G″ crossover. These features areindicative of “entanglement network” materials (Clark A. and S. B.Ross-Murphy, “Structural and Mechanical Properties of Biopolymer Gels.Adv. Poly. Sci. Springer-Verlag, Berlin, Heidelberg, 1987).

As stated hereinabove, the therapeutic composition of the invention iscapable of maintaining a liquid state in storage at room temperature(e.g., at about 24-25° C.) for at least 24 hr. Preferably, thecomposition is capable of maintaining a liquid state at room temperaturefor at least 48 hr, more preferably for at least seven days. Whenrefrigerated (e.g., at about 4-8° C.) the composition of the inventionis preferably capable of maintaining a liquid state for a period of atleast one month. Accordingly, the composition of the invention includesa network of a viscoelastic material which is stably maintained in asolution state.

As a solution, the composition can be administered into a body tissuevia a surgical needle such as an 18-27 gauge bore needle, as furtherdescribed in details in Examples 2-5 and 7-8 hereinbelow.

Surprisingly and unexpectedly, the present inventors uncovered that thecomposition of the invention can be delivered into a damaged myocardiumvia intracoronary administration (see Example 6 hereinbelow). Thesefindings indicate that the composition is uniquely capable of flowingwithin a blood vessel, crossing out of blood capillaries and spreadinginto the extracellular matrix of the surrounding tissue.

As used herein, the phrase “blood vessel” refers to an artery, a vein ora capillary.

Following deposition within a body tissue, the composition of theinvention assumes a gel state. The transition from a liquid to gel stateresults from the diffusion of water from the viscoelastic matrix intothe surrounding extracellular medium.

Once gelatinized, the viscoelastic material provides substantialmechanical support and elasticity to the body tissue, as well asscaffolding for new tissue regeneration.

Hence, the capacity of the cross-linked polymer solution to assume a gelstate, following deposition in the target body tissue, is an essentialfeature of the present invention.

Optionally, the therapeutic composition of the invention furtherincludes cells and/or at least one therapeutic agent.

Suitable cells which may be included in the composition of the present 5invention can be, for example, caidiomycetes, myoblasts, fibroblasts,chondrocytes, muscle cells, smooth muscle cells, endothelial cells,mesenchymal cells and embryonic stem cells. The cells can be mixed withthe cross-linked polymer described hereinabove to make the therapeuticcomposition of the present invention.

Suitable therapeutic agents which may be included in the composition ofthe present invention can be, for example, growth factors (e.g., basicfibroblast growth factor; bFGF), vascular endothelial growth factor(VEGF), insulin-like growth factor (IGF), members of the TGF-family,bone morphogenic proteins (BMP), platelet. derived growth factors,angiopoietins, and other factors such as myogenic factors, transcriptionfactors, cytokines, and homeobox gene products, polynucleotides,polypeptides, hormones, anti-inflammatory drugs, anti-apoptotic drugs orantibiotic drugs.

Optionally, the therapeutic agent or agents can be chemically linked tothe polymer of the invention. Such linkage can be effected via any knownchemical bonding approach, preferably a covalent bond. A suitablecovalent bond can be, for example, an ester bond (e.g., a carboxylicester bond, an oxyalkyl carboxylic ester bond, an amide bond, or athioester bond), a glycosidic bond, a carbonate bond, a carbamate bond,a thiocarbamate bond, a urea bond or a thiourea bond.

Therapeutic compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration: The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the therapeutic compositions or human orveterinary administration. Such notice, for example, may be of labelingapproved by the U.S. Food and Drug Administration for medical devices ordrugs, or of an approved product insert. Therapeutic compositions mayalso be prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition, as if further detailed above.

Optionally, the therapeutic composition of the invention may be packagedin a kit, along with a devise suitable for administering the therapeuticcomposition into a body tissue and with a packaging material identifyingthe kit for use in treatment of the body tissue.

As mentioned hereinabove, the present inventors unexpectedly uncoveredthat providing cross-linked alginate solution into infarcted myocardiumtissues of rats and to pigs, via injection or intra-coronaryadministration, substantially improved the heart function; inducedangiogenesis and promoted regeneration of the damaged tissue (seedetails in Examples 2-6 and 8 hereinbelow). In addition, thecross-linked alginate solution was shown capable of substantiallyimproving blood flow in ischemic limb tissues of rats (Example 7).

Thus, according to another aspect of the present invention there isprovided a method of treating a body tissue, such as a damaged bodytissue, by providing the body tissue with an effective amount of thetherapeutic composition of the present invention.

The term “treatment” used herein encompasses the complete range oftherapeutically positive effects of administrating the composition ofthe present invention to a body tissue, including improving the tissuefunction, providing mechanical support and promoting tissue healing andrepair processes (e.g., angiogenesis and new tissue regeneration). Theterm treatment further includes reduction of, alleviation of, and reliefof diseases or disorders associated with a damaged tissue. In addition,the term treatment includes prevention or postponement of development ofdiseases or disorders associated with a damaged tissue.

The phrase “body tissue” used herein encompasses any mammalian bodytissue, preferably a human body tissue. A body tissue, according to theteachings to the present invention, can be, but not limited to, amyocardial tissue, a muscle tissue, a kidney tissue, a cartilage tissue,a bone tissue, or a dermal tissue.

The phrase “damaged body tissue” used herein encompasses any body tissuewhich is functionally and/or structurally impaired, such as, but notlimited to, an infarcted (post MI) myocardium, an ischemic myocardium,an ischemic muscle, an ischemic cartilage, an ischemic bone or anischemic dermis.

The term “angiogenesis” used herein refers to the process ofvascularization of a tissue involving the development of new capillaryblood vessels.

As mentioned hereinabove, the therapeutic composition of the presentinvention can be injected directly into a damaged body tissue or beadministered intravenously.

Advantageously, the therapeutic composition of the present invention canbe administered intra-arterially, preferably intra-coronarily, via acatheter such as described, for example, by Knight et al. (Circulation95:2075-2081, 1997). Most preferably, the composition is administeredintra-coronarily via an over-the-wire balloon catheter such asdescribed, for example, in Examples 6 hereinbelow.

The phrase “effective amount” used herein refers to an amount effectiveto provide a significant therapeutic benefit. The amount of acomposition to be administered will, of course, be dependent on thesubject being treated, the severity of the affliction, the manner ofadministration, the judgment of the prescribing physician, etc.Preferably, an effective amount ranges between 0.1 and 10 ml (dwtbetween 1 and 10 mg), more preferably between 0.5 and 5 ml (dwt between5 and 50 mg), most preferably between 1 and 4 ml (dwt between 10 and 40mg).

The composition of the present invention can also be used to effectivelytreat chronic heart conditions such as, for example, congestive heartfailure (CHF) and ischemic mitral regurgitation (MR), by providing theheart tissue with an effective amount of the therapeutic composition.

The composition of the present invention can be used to induceangiogenesis in a damaged heart tissue, by providing the damaged hearttissue with an effective amount of the therapeutic composition.

In addition, the composition of the present invention can be used toinduce angiogenesis and promote blood flow in an ischemic muscle tissue,such as an ischemic limb muscle tissue, by providing the damaged muscletissue with an effective amount of the therapeutic composition.

Hence, the present invention provides a novel aqueous cross-linkedpolymer solution which can be delivered into a body tissue via injectionor intra-arterial administration, so as to provide substantialtherapeutic benefits to the treated body tissue effectively and safely.

Although the detailed descriptions above relate primarily to alginatesolutions, it should be noted that that the teachings of the presentinvention can be applied to solutions of other biocompatible polymers(e.g., chitosan, gellan gum, carageenan, polyphosphazines,polyacrylates), since parameters governing preparation of thecross-linked alginate solutions described above (e.g., exhibiting anelastic response which is equal to or greater than its viscous responseunder small deformation oscillatory frequencies in the linearviscoelastic limit and a shear thinning behavior in a power-lawrelationship) can be used as guidelines for preparing other types ofcross-linked polymer solutions capable of maintaining a liquid state instorage at room temperature and assuming a gel state followingdeposition within the body tissue.

As used herein, the term “about” denotes ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used• in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Example 1 Preparation and Rheological Evaluation of Cross-LinkedAlginate Solutions Materials and Methods: Preparation of Sodium Alginateand Calcium Gluconate Mixtures

Sodium alginate samples (Mw ranging from about 15 to 160 kDa; G/M ratio2.1) were purchased from FMC Biopolymers, Orammen, Norway. Sodiumalginate samples were dissolved in double distilled water (DDW) to afinal concentration of 2% (w/v) then mixed with 2% (w/v) calciumgluconate solution (D-gluconic acid, hemi-calcium salt, Sigma). Themixtures were homogenized using Heidolph DIAX 900 homogenizer equippedwith 10G head, operating at 26,000 rpm for about 2 minutes. Followinghomogenization, the preparations were refrigerated at about 4-8° C.until use.

Alginate Molecular Weight Determination

Sodium alginate samples were separated on a chromatographic systemcomprising a Waters 606 pump followed by two PSS Suprema gel permeationcolumns connected in a series. Column dimensions were 300×8 mm²,particle size 10 mm, porosity of 3000 and 10,000 A. Flow rate was 0.5ml/min. The columns were kept at a constant temperature of 25° C. insidea Techlab K-4 controlled oven. The chromatographic system was attachedto a Dawn DSP (Wyatt Technology Corporation) multi-angle laser lightscattering (MALLS) photometer equipped with a He/Ne laser working at632.8 nm, a K5 refraction cell and 18 detectors at angles 14-1630.Concentration was monitored by a calibrated interferometricrefractometer Optilab DSP (Wyatt Technology Corporation). Dataprocessing and molar mass calculation were performed with Wyatt ASTRAsoftware version 4.7. Each sample was injected three times to ensurereproducibility. The alginate dn/dc was estimated (using Optilab OSP,controlled by Wyatt dn/dc software) to be 0.155 ml/g (aqueous buffer).Aqueous buffer solutions were prepared from ultra pure water (0.055μs/cm; USF SERAL Purelab RO75 followed by USF SERAL Purelab UV)supplemented with 0.1 M NaNO₃, 0.02% (w/v) NaN₃ and 10 mM imidazole. Thebuffer was titrated with NaNO₃ to pH 7.0 and filtered through a 0.1 μmfilter (Gelman Sciences VacuCap 60).

Rheological Analysis

Rheological analyses were made using a CarriMed CLS50 controlled stressrheometer (CarriMed Instruments Ltd. Dorking, UK) operated in thecone-plate mode (cone angle 1° and 4°, having 60 and 40 mm diameter,respectively). Small amplitude oscillatory shear experiments (0.1-10 Hz)were performed within the linear viscoelastic limit. Frequency scanswere performed at the lowest stress possible to prevent damage to thesample. The linearity of the response was monitored continuously toascertain linear viscoelasticity.

Results:

The molecular weight (Mw) and polydispersity (PD; a measure of Mwdistribution range) values of selected sodium alginate samples, asdetermined by GPC-MALLS analysis, are shown in Table 1 below.

TABLE 1 Table 1: Molecular characteristics of different atginatesApproximate Mn Polydispersity Average Mw Alginate type (g/mol) Mw(g/mol) (Mw/Mn) (kDa) LF 5/60* 1.348e+4  1.68+4 1.246 ± 0.001 151.353e+4 1.639e+4 1.212 ± 0.002 LF 5/60 2.102e+4 2.752e+4 1.309 ± 0.00730 2.113e+4 2.656e+4 1.257 ± 0.01  LVG 8.986e+4 1.029e+5 1.145 + 0.006100 9.038e+4 9.877e+4 1.093 + 0.002 LVG 1.469e+5 1.667e+5 1.135 ± 0.016160 1.385e+5 1.559e+5 1.126 ± 0.012 MVG 2.103e+5 2.596e+5 1.235 ± 0.006250 2.055e+5 2.385e+5 1.161 ± 0.007 *alginate was subjected to acontrolled degradation.

Plots of viscosity vs. shear rate of sodium alginate solutions exhibitedessentially constant viscosity for all shear rates. The average measuredviscosity values of the sodium alginate solutions are shown in Table 2below.

TABLE 2 Table 2: Viscosity as a function of shear rate (10⁻²-10⁴/sec)Alginate Solution Viscosity (cP) 1% (w/v) LF 5/60 40 1% (w/v) LVG 127 1%(w/v) MVG 400

As can be seen in FIGS. 1A-B and 2, providing sodium alginate solutionswith calcium ions resulted in decreasing of the solutions viscosity byincreasing shear rates. Such behavior, also known as a shear thinning(or pseudoelastic) behavior in a power-law relationship, is indicativeof a structured material (Lapasin and Pricl, “Rheology of IndustrialPolysaccharides: Theory and Application,” London, Blackbie, 620 pp.,1995).

Viscoelastic spectra of various preparations (exhibiting changes ofviscosity, elastic response and viscous response values under increasingoscillatory frequencies) are shown in FIGS. 3-4 and 15-34.

As can be seen in FIGS. 3A, 4A, 10, 18, 21 24, 28 and 30 and in Table 3hereinbelow, sodium alginate solutions devoid of calcium ions exhibitedviscous response (G″) greater than the elastic response (G′) underoscillatory frequencies in the linear viscoelastic limit (0.1-10 Hz).Such material behavior is indicative of a random coil polymer solution(i.e., having no polymer cross-linking).

As can be seen in FIGS. 11-14, 19, 22, 26, 29 and 31 and in Table 3hereinbelow, sodium alginate solutions mixed with calcium gluconate at aratio being over 1:0.2, respectively, also exhibited viscous response(G″) greater than the elastic response (G′) under oscillatoryfrequencies in the linear viscoelastic limit. These results indicatethat the level of polymer cross-linking in these preparations wasnegligible.

On the other hand, mixtures of sodium alginate and calcium gluconate ata ratio being under 1:0.5, respectively, formed gels due to substantialpolymer cross linking.

Unexpectedly, mixtures of sodium alginate and calcium gluconate at aratio ranges between about 1:0.4 to 1:0.3 developed into stablesolutions which remained freely flowable for at least 24 hr at roomtemperature and for at least 30 days at 4-8° C. Mechanical spectra ofthese novel solutions show a crossover between the viscous response andthe elastic response values (FIGS. 3B-C, 4B, 15, 16, 17, 20, 23, 27 and33 and in Table 3 hereinbelow). Such behavior is typical of an“entanglement network” viscoelastic material (Clark, A. and Ross Murphy,S. B., “Structural and Mechanical Properties of Biopolymer Gels.” Adv.Poly. Sci. Springer-Verlag, Berlin, Heidelberg, 1987).

TABLE 3 Table 3: Visual appearance and rheological characteristics ofhomogenized mixtures of sodium alginate and calcium gluconate SodiumCalcium Alginate Alginate Gluconate Average Concentration ConcentrationVisual Rheological Mw (kDa) (% w/v) (% w/v) Appearance Characteristics*15 1 0 Flowable solution G″ > G′ 15 1 0.2 Flowable solution G″ > G′ 15 10.3 Flowable solution G″ > G′ 15 1 0.4 Flowable solution G′ ≧ G″ 15 10.5 Gel — 30 1 0 Flowable solution G″ > G′ 30 1 0.2 Flowable solutionG″ > G′ 30 1 0.3 Flowable solution G′ ≧ G″ 30 1 0.4 Gel — 30 1 0.5 Gel —30 0.8 0 Flowable solution G″ > G′ 30 0.8 0.2 Flowable solution G″ > G′30 0.8 0.3 Flowable solution G′ ≧ G″ 30 0.8 0.4 Gel — 30 0.8 0.5 Gel —30 1.5 0 Flowable solution G″ > G′ 30 1.5 0.2 Flowable solution G″ > G′30 1.5 0.3 Flowable solution G′ ≧ G″ 30 1.5 0.4 Gel — 30 0.5 0.5 Gel —100 1 0 Flowable solution G″ > G′ 100 1 0.1 Flowable solution G″ > G′100 1 0.2 Flowable solution G″ > G′ 100 1 0.23 Flowable solution G″ > G′100 1 0.25 Flowable solution G″ > G′ 100 1 0.270 Flowable solution G′ ≧G″ 100 1 0.275 Flowable solution G′ ≧ G″ 100 1 0.3 Flowable solution G′≧ G″ 100 1 0.35 Gel — 100 1 0.4 Gel — 160 1 0 Flowable solution G″ > G′160 1 0.2 Flowable solution G″ > G′ 160 1 0.3 Gel — 160 1 0.4 Gel — 1601 0.5 Gel — *G′ = elastic response; G″ = viscous response.

Hence, the results indicate that stable cross-linked alginate solutionscan be produced by homogenizing mixtures of sodium alginate and calciumgluconate solutions. The novel cross-linked alginate solutions arefreely flowable and exhibit elastic responses which are equal to orgreater than their viscous response under small deformation oscillatoryfrequencies in the linear viscoelastic limit.

Example 2 Injection of Cross-Linked Alginate Solution into a RatInfarcted Myocardium Materials and Methods: Aqueous Cross-LinkedAlginate Solution

The solution was made of 1% w/v sodium alginate (Avg. Mw=30 kDa; G/Mratio 2.1) and 0.3% w/v calcium gluconate, as described in Example 1hereinabove.

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Induction of Myocardial Infarction

Male Sprague-Dawley rats (about 250 g) were anesthetized with acombination of 40 mg/kg ketamine and 10 mg/kg xylazine, intubated andmechanically ventilated. The rat chest was opened by left thoracotomy,the pericardium was removed and the proximal left coronary arterypermanently occluded with an intramural stitch.

Injection of the Cross-Linked Alginate Solution

One week following MI, animals were anesthetized and their chest wasopened under sterile conditions. The infarcted myocardial area wasvisually recognized based on the appearance of surface scar and wallmotion akinesis. The aqueous cross-linked alginate solution or a serumfree culture medium (control) were injected (100-200 μL volume) into thescar tissue using a 27-gauge needle. Following injection the surgicalincision was sutured closed.

Echocardiography

Transthoracic echocardiography was performed on all animals within 24hours post MI (baseline echocardiogram) and eight weeks• followinginjection using the procedure described by Etzion et al. (1. Mol. CellCardiol. 33: 1321-1330, 2001) and 30 Leor et al. (Circulation102:III56-61, 2000). The measured parameters were: LV anterior wallthickness; maximal LV end-diastolic dimension; minimal left ventricularend-systolic dimension in M-mode and 2-D imaging; and fractionalshortening (as a measure of systolic function) calculated as[FS(%)=(LVIDd−LVIDs)/LVIDd×100], where LVID indicates LV internaldimension, s is systole, and d is diastole. Index of change in LV area(%) was calculated as [(EDA−ESA)/EDA]×100 where EDA indicates LV enddiastolic area, ESA indicates LY end systolic area (Mehta et al., J. Am.Coll. Cardiol. 11:630-636, 1988). All measurements were averaged forthree consecutive cardiac cycles.

Histological Examination

Eight weeks following injection, animals were sacrificed with anoverdose of phenobarbital. Hearts were harvested, and processed forhistological and immunohistochemical examination. Adjacent blocks wereembedded in paraffin, sectioned (5 μm thickness), stained withhematoxylin and eosin then with labeled antibodies against α-actinsmooth muscle (SMA; (SIGMA), fast myosin heavy chain (MHC; Sigma), ED1(Dako), proliferating cell nuclear antigen (PCNA; Enco) and SDF-1 (R&Dsystems).

Angiogenesis Assessment

The effect of alginate injection upon neoangiogenesis(neovascularization) in the infarcted and peri-infarcted myocardium wasassessed by immunohistologic staining of representative slides usinganti-αSMA antibody (Sigma) to pericytes and arterioles. Followingpreliminary microscopic examination under low power, five consecutiveadjacent fields were photographed from each section at a magnificationof ×200. The number of vessels was assessed from photomicrographs bycomputerized image analysis to count the number of vessels and tocalculate vessel density (mean number of capillaries and arterioles/mm²)in the myocardium of treated and control animals.

Statistical Analysis

Univariate differences between the control and treated groups wereanalyzed using t tests for continuous variables. Changes betweenbaseline and eight week data were analyzed using paired t tests.Comparisons of the changes between baseline and eight week data wereanalyzed by repeated-measures ANDYA using GraphPad Prism version 4.00for Windows (GraphPad Software, San Diego, Calif., USA). The ANDVA modelincluded the control versus treated and baseline versus eight week asfactors and the interaction between the two factors (Perin et al.,Circulation; 107:2294-2302, 2003). A probability value p≦0.05 wasconsidered statistically significant.

Results:

In one experiment, seven days post MI, rats were treated with eithercross-linked alginate solution, embryonic cardiomyocyte suspension(1.5×10⁶ cells; cells control) or a serum free culture medium (mediumcontrol). 2-D echocardiography results (Table 4) show that thecross-linked alginate solution per se increased scar thickness,increased cardiac contractility and reduced LV dilatation anddysfunction, as compared with the medium control.

TABLE 4 Table 4: 2-D echocardiography analyses Treatment¹ (n = 8) CellsControl (n = 5) Meadium Control (n = 4) Pre 8 wk Post Pre 8 wk Post Pre8 wk Post ANOVA injection Injection P value injection Injection P valueinjection Injection P value P value AW (d cm) 0.12 ± 0.01 0.15 ± 0.010.03  0.11 ± 0.004 0.14 ± 0.01 0.01  0.12 ± 0.005 0.11 ± 0.01 0.9 0.3LVEDD (cm) 0.71 ± 0.02 0.86 ± 0.06 0.07 0.68 ± 0.03 0.69 ± 0.02 0.7 0.73± 0.04 0.97 ± 0.04 0.003 0.01 LVESD (cm) 0.52 ± 0.05 0.65 ± 0.07 0.10.45 ± 0.03 0.45 ± 0.04 0.9 0.56 ± 0.05 0.81 ± 0.04 0.004 0.01 LVED area 0.4 ± 0.03  0.6 ± 0.07 0.03 0.40 ± 0.04 0.40 ± 0.01 1 0.41 ± 0.06 0.66± 0.05 0.007 0.06 cm² LVES area 0.2 ± 0.4  0.3 ± 0.06 0.1 0.17 ± 0.020.19 ± 0.03 0.7 0.24 ± 0.04 0.43 ± 0.04 0.009 0.05 cm² LV FS (%) 27 ± 5 25 ± 4  0.6 34 ± 3  35 ± 6  0.8 24 ± 3  17 ± 1  0.06 0.06 FAC (%) 44 ±4  44 ± 5  1 57 ± 2  54 ± 6  0.6 42 ± 2  36 ± 1  0.14 0.08¹Treatment—cross linked alginate solution ²Cells control—fetalcardiomycetes suspended (1 × 10⁶ cells) in culture medium ³Mediumcontrol—culture medium only AW d—Anteriour wall diastolic thicknessLVEDD—LV end diastolic dimension LVESD—LV end systolic dimension LV EDarea—LV end diastolic area LV ES area—LV end systolic area LV FS—LVfractional shortening - [(LVIDd − LVIDs)/LVIDd] × 100 FAC %—Fractionalarea change -[(EDA − ESA)/EDA] × 100

In another experiment, rats the cross-linked alginate solution or with aserum free culture medium was injected into rat myocardial scar 7 dayspost MI. In addition, two rats with normal heart were treated with thecross-linked alginate solution.

Echocardiography results show significant increases in LV diastolic andsystolic internal diameters were observed in control animals, whichindicate extensive myocardial infarction, LV remodeling and heartfailure (FIGS. 5,6; Table 5). In addition, the LV end-diastolic andsystolic cavity areas increased by 75% and by over 100%, respectively,in the control animals (FIGS. 5, 6, Table 5; p<0.05). These results areconsistent with the process observed in human patients followingextensive anterior MI (Pilla et al., Circulation 106:1207-211, 2002).Progressive LV dilatation from baseline was also accompanied bysignificant deterioration in LV performance, reflected by thedeterioration of fractional shortening (from 30±5% at baseline to 22±3%;p<0.05) and percentage of LV fractional area change (from 49±5% to38±3%; p<0.05; FIG. 7).

On the other hand, the cross-linked alginate solution significantlyincreased the scar thickness (p<0.0001) and attenuated the typicalcourse of LV dilation complicating extensive anterior MI (FIG. 5; Table5). Furthermore, the cross-linked alginate solution substantiallyreduced the LV diastolic and systolic dimensions, as compared with thecontrol animals (FIG. 5; Table 5).

TABLE 5 Table 5: 2-D and Doppler echocardiography analyses Treatment¹ (n= 15) Control² (n = 9) Treatment Pre 8 wk Post Pre 8 wk Post vs. Controlinjection Injection P value injection Injection P value P value AW (dcm) 0.14 ± 0.01 0.16 ± 0.01 <0.001 0.14 ± 0.01 0.14 ± 0.01 0.8 0.16LVEDD (cm) 0.71 ± 0.02 0.86 ± 0.03 <0.01 0.73 ± 0.02 0.98 ± 0.03 <0.00010.02 LVESD (cm) 0.50 ± 0.03 0.65 ± 0.04 <0.01 0.51 ± 0.04 0.78 ± 0.05<0.0001 0.05 LVED area 0.38 ± 0.03 0.56 ± 0.05 0.17 0.40 ± 0.03 0.70 ±0.03 <0.0001 0.05 cm² LVES area 0.21 ± 0.03 0.32 ± 0.04 0.05 0.20 ± 0.020.44 ± 0.04 <0.0001 0.06 cm² LV FS (%) 29 ± 3  25 ± 2  0.2 30 ± 5  20 ±3  0.02 0.3 FAC (%) 47 ± 4  45 ± 3  0.6 49 ± 4  38 ± 3  0.02 0.1¹Treatment—cross linked alginate solution ²Control—culture medium AWd—Anteriour wall diastolic thickness LVEDD—LV end diastolic dimensionLVESD—LV end systolic dimension LV ED area—LV end diastolic area LV ESarea—LV end systolic area LV FS—LV fractional shortening - [(LVIDd −LVIDs)/LVIDd] × 100 FAC %—Fractional area change -[(EDA − ESA)/EDA] ×100

Immunostaining with anti α-SMA antibody indicates increasedneoangiogenesis and migration of myofibroblasts in myocardium which hadbeen treated with the cross-linked alginate solution, as compared withuntreated control. The blood vessel density values in treated andcontrol myocardium were 231±13 and 180±16, respectively (p<0.02; FIG.8).

Immunostaining with anti-PCNA antibody indicates DNA activity andproliferation of endothelial cells and cardiomyocytes in the treatedmyocardium.

Hence, these results indicate that the cross-linked alginate solution ofthe present invention can substantially improve the heart function ofrats following MI and promote angiogenesis and regeneration of thedamaged myocardium.

Example 3 Injection of Cross-Linked Alginate Solution into a PigInfarcted Myocardium Materials and Methods: Aqueous Cross-LinkedAlginate Solution

The solution was made of 1% w/v sodium alginate (Avg. Mw=30 kDa; G/Mratio 2.1) and 0.3% w/v calcium gluconate, as described in Example 1hereinabove.

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Induction of Myocardial Infarct (MI)

Myocardial infarct was induced according to Yau et al. (Ann Thorac Surg75:169-176, 2003) and Watanabe et al. (Cell Transplant 7:239-246, 1998).Briefly, female Sincklaire (mini) pigs weighing 30-40 kg werepre-medicated with ketamine (20 to 30 mg/kg, intramuscular) beforeanesthetic induction with 4% isoflurane. Anesthesia was maintained withisoflurane 1% to 2.5%. The right femoral artery was isolated andcannulated with an introduction sheath. Through this, a cardiac catheterwas placed in the mid portion of the left anterior descending artery(LAD) and an embolization coil (Boston Scientific, USA) was extrudedfrom the catheter with a guide wire and placed in the distal portion ofLAD under fluoroscopic guidance. This procedure induced a thrombusresulting in myocardial infarction in the left ventricle, which wasconfirmed by angiography and electrocardiography. Electrical DCcardioversion was given as was necessary.

Induction of Mitral Regurgitation (MR)

Mitral regurgitation was produced by creating extensive posterior MIfollowing coil embolization of the circumflex coronary artery.

Injection of the Cross-Linked Alginate Solution

For evaluating the effect of the cross-linked alginate solution on MI,four animals were subjected to anterior MI. Five of these animals wereinjected with the cross-linked alginate solution and the other fiveanimals were injected with saline (control). For evaluating the effectof cross-linked alginate solution on MR, four animals were subjected topostero-lateral MI. Two of these animals were injected with thecross-linked alginate solution and the other two animals were injectedwith saline (control).

Myocardium injections were performed 7 to 10 days post MI as follows:animals were anesthetized, their chest was opened and the infarcted areawas identified visually by surface scar and wall motion abnormality.Aliquots (about 2.5 ml) of the cross-linked alginate solution, or salinecontrol, were injected into the infracted myocardium. Followinginjection, air was expelled from the chest and the surgical incisionsutured closed. Eight weeks following treatment, all surviving animalswere euthanized with overdosed Phenobarbital then their hearts wereharvested and processed for histological analyses.

Echocardiography

Ecocardiogram analysis was performed immediately following MI prior toinjection treatment, as well as on day 10, 30 and 60 following MI. Theanalyses were performed using a phased-array transducer (2.5 MHz)equipped with an ultrasound system (Sonos 5500, Hewlett-Packard,Andover, Mass.). Images were recorded on VHS videotape. End-diastolicand end-systolic frames were selected from standard apical andparasternal views.

Global LV ejection fraction (LVEF) was estimated visually. LV volumeswere measured by manually tracing the left ventricular cavity using thesingle plane modified Simpson's algorithm when >80% of the endocardialborder could be detected in both the apical 4- and 2-chamber views, andby a single plane when 80% of the endocardial border could be detectedonly in the apical 4-chamber view.

Regional myocardial assessment and wall motion score index values weredetermined by assigning a segmental score (1=normal, 2=hypokinetic,3=akinetic, 4=dyskinetic) to each of the 16 left ventricular segments,as recommended by the American Society of Echocardiography (Schiller etal., J. Am. Soc. Echocardiogr. 2:358-367, 1989). All segment scores wereadded and divided by the number of segments analyzed to obtain the wallmotion score index. Left ventricular wall motion score index (WMSI) wasderived using the sum of the individual scores divided by the totalnumber of analyzed segments. Regional motion score index was calculatedby the same method for the segments of the mid-LAD territory (infarctrelated artery territory). The data were interpreted by a singleexperienced observer, and all measurements were obtained off line by asingle technician.

Mytral regurgitation (MR) was graded by color Doppler flow mapping usingan algorithm which integrated jet expansion within the left atrium jeteccentricity. The size of the proximal area. MR was considered mild whenthe regurgitant jet area was under 20% of the LA area (in the absence ofa wall jet and a proximal isovelocity surface area visible withoutbaseline shifting). MR was considered severe when regurgitant jet areaoccupied was over 40% of the LA area. Jet eccentricity or a sizableproximal flow convergence radius (0.6 mm in a patient with jet areaunder 20%, or 0.9 mm in a patient with a jet area between 20% and 40%)raised the grade of MR by one degree.

Morphological and Histological Examination

Animal hearts were arrested with potassium chloride and rapidly excised.Their atria were removed and the coronary arteries were perfused with100 mL 10% formaldehyde. The myocardium was fixed in diastole with anintraventricular pressure (30 mm Hg) in formaldehyde solution. Followingfixation, the myocardium was sliced (5 mm thick) and each section wasphotographed. The mean scar length was calculated as the mean of theepicardial and the endocardial scar lengths. The scar area wascalculated as the mean scar length multiplied by 0.5 cm. The total scararea was calculated as the sum of scar areas of all sections combinedand the scar volume was calculated as total scar area multiplied by themean scar thickness. A cube of tissue from the center of the infarctzone measuring about 5 mm³ was embedded in paraffin and cut into 5 μmsections for staining with hematoxylin and eosin.

For immunohistochemical examination, tissue slices were seriallyrehydrated in 100%, 95%, and 70% ethanol after deparaffinization withtoluene. Endogenous peroxidase in the sample was blocked and the sampleswere stained with antibodies. Adjacent blocks were embedded in paraffin,sectioned into 5 μm slices and stained with hematoxylin and eosin.Serial sections were immunolabelled with antibodies against SMA, slowMHC (Sigma), Ki67 (Novocastra Ltd.) and SDF-1 (R&D systems).

Statistical Analysis

Univariate differences between the control and treated groups wereanalyzed using t tests for continuous variables. Changes betweenbaseline and eight week data were analyzed using paired t tests.Comparisons of the changes between baseline and eight week data wereanalyzed by repeated-measures ANOVA using GraphPad Prism version 4.00for Windows (GraphPad Software, San Diego, Calif., USA). The ANOVA modelincluded the control versus treated and baseline versus eight week asfactors and the interaction between the two factors (perin et al.,Circulation; 107:2294-2302, 2003). A probability value p≦0.05 wasconsidered statistically significant.

Results:

During 60 days post MI two out of five animals of the control groupsdied (3 and 49 days post MD. All of the five treated animals survivedthe 60 days period post MI.

The treated myocardium exhibited high density of endothelial cells andcardiomyocytes which reacted with anti-Ki67 antibody (indicative of DNAactivity). In contrast, the control myocardium exhibited high density ofcells having fibroblast morphology; while reactivity with Ki67 wasobserved within the infarct zone only. These observations indicate thatthe cross-linked alginate solution is capable of inducing proliferationof endothelial cells and cardiomyocytes and, consequently, cardiacregeneration.

As illustrated in FIGS. 9A and B, treated myocardium which hadcircumflex occlusion exhibited repositioning of papillary muscles towardthe anterior annulus. These observations show that providing thecross-linked alginate solution into the myocardium can reduce ischemicMR without compromising LV function.

Hence, these results indicate that the cross-linked alginate solution ofthe present invention can improve survival of pigs following MI and canpromote regeneration of the damaged myocardia.

Example 4 Injection of Cross-Linked Alginate Solution Combined withMyoblasts into a Rat Normal Myocardium Materials and Methods: AqueousCross-Linked Alginate Solution

The solution was made of 1% w/v sodium alginate (Avg. Mw=30 kDa; G/Mratio 2.1) and 0.3% w/v calcium gluconate, as described in Example 1hereinabove.

Skeletal Myoblasts

Myoblasts from the hind limb muscle of Sprague-Dawley neonatal rats wereisolated and purified according to the procedure described byRosenblatt, J. D. (In Vitro 20 Cell Dev. Biol. Anim. 31:773-779, 1995).

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel-Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Injection

Male Sprague-Dawley rats (about 250 g) were anesthetized with acombination of 40 mg/kg ketamine and 10 mg/kg xylazine, intubated andmechanically ventilated. The rat chest was opened by left thoracotomy,the pericardium was •removed and the rats were subjected to injection,using a 27-gauge needle, with 100-200 μL of a suspension of skeletalmyoblasts in the cross-linked alginate solution; or with a suspension ofskeletal myoblasts in a serum free culture medium (control). Followinginjection, the surgical incision was sutured closed.

Histological and Immuno-Histological Examination

Four weeks following injection, animals were sacrificed with an overdoseof phenobarbital. Hearts were harvested, and processed for histologicalexamination. Adjacent blocks were embedded in paraffin, sectioned into 5μm slices and stained with hematoxylin and eosin. Serial sections wereimmunolabelled with antibodies against fast MHC (Sigma).

Results:

Injection of skeletal myoblasts combined with cross-linked alginatesolution neoangiogenesis and neovascularization, as shown by theformation of functional new vessels, which can be evidenced by thepresence of red blood cells. Furthermore, the treatment substantiallyincreased the retention of transplanted myoblasts at the injection site,as compared with the control.

Example 5 Injection of Cross-Linked Alginate Solution Combined withHuman Blood-Derived Progenitor Cells into a Rat Infarcted MyocardiumMaterials and Methods: Aqueous Cross-Linked Alginate Solution

The solution was made of 1% w/v sodium alginate (Avg. Mw=30 kDa; G/Mratio 2.1) and 0.3% w/v calcium gluconate, as described in Example 1hereinabove.

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel-Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Induction of Myocardial Infarction

Athymic nude rats (about 250 g) were anesthetized using a mixture of 40mg/kg ketamine and 10 mg/kg xylazine, then intubated and mechanicallyventilated. The animal chest was opened using left thoracotomy, thepericardium was removed and the proximal left coronary arterypermanently occluded with an intramural stitch.

Injection of the Cross-Linked Alginate Solution

One week post MI animals chest was opened and the infarcted area wasidentified visually on the basis of surface scar and wall motionakinesis. Aqueous cross-linked alginate solution (100-200 μL), or aserum free culture medium (control) was injected into the myocardialscar tissue using a 27-gauge needle. Following injection the surgicalincision was sutured closed.

Infusion of Human Blood-Derived Progenitor Cells

One week following injection animals were treated with intravenousinfusion of human blood-derived CD 133⁺ progenitor cells (2-4×10⁶cells).

Histological and Immuno-Histological Examination

One week following infusion animals were sacrificed by administering anoverdose of phenobarbital. Hearts were harvested and processed forhistological examination. The presence of human donor cells in recipientrat myocardium was confirmed by immunostaining using anti-HLA-DRantibodies.

Results:

Infused donor cells (highlighted brown color) colonized the scar tissueat site of injection of the cross-linked alginate solution. Thesehistological observations indicate that the cross-linked alginatesolution is capable of effectively promoting homing of stem orprogenitor cells to the injected site.

Example 6 Intra-Coronary Administration of Cross-Linked AlginateSolution to Pig Infarcted Myocardium Materials and Methods: Preparationof Biotinylated Alginate

Sodium alginate solution (2% w/v; Avg. Mw=30 kDa; GIM ratio 2.1) wasdiluted in an equal volume of 1 M 2-[N-morpholino]ethansulfonicacidmonohydrate (MES) buffer at pH 6.0 [resulting in 1% (w/v) alginate in0.5 M MES]. Biotin hydrazide (0.052 g; 0.2 mmol) was added to thealginate solution (5 ml) and the mixture was stirred to yield a stablesuspension. The suspension was then supplemented withN-hydroxysulfosuccinimide (NHSS; 0.0217 g; 0.1 mmo)) and1ethyl-3-(dimethyaminopropyl)carbodimide HCl (EDAC; 0.0384 g; 0.2 mmol)and stirred at room temperature for 2 hr. The resulting product wasdialyzed against 2 liter DDW using 8000 MWCO membrane (water was changedtwice a day for two days) then lyophilized.

Aqueous Cross-Linked Alginate Solution

The cross-linked alginate solution was made of biotinylated ornon-biotinylated sodium alginate (1% w/v; Avg. Mw=30 kDa; GIM ratio 2.1)and calcium gluconate (0.3% w/v) using the procedure described inExample 1 hereinabove.

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Induction of Myocardial Infarction (Ml)

Myocardial infarction was induced according to Yau et al. (Ann ThoracSurg 75:169-176, 2003) and Watanabe et al. (Cell Transplant 7:239-246,1998). Briefly, female domestic pigs weighing 50-75 kg wereintramuscularly pre-medicated with ketamine (20 to 30 mg/kg), thenanesthetized using 4% isoflurane. Anesthesia was maintained withisoflurane 1% to 2.5%. MI was induced via cardiac catheterization andinflation of coronary angioplasty balloon in the left anteriordescending (LAD) coronary artery (beyond the first diagonal branch toocclude LAD flow) for 60 or 90 minutes. This occlusion resulted inmyocardial infarction in the left ventricle which was confirmed byangiography, electrocardiography and echocardiography analyses. Severeventricular arrhythmias appearing during occlusion were treated byintravenous administration of amiodarone or lidocaine. Followingocclusion, animals were administered intravenously with heparin (5,000VI) and aspirin (250 mg) then allowed to recover. Analgesics wereadministered intramuscularly for pain control. Antibiotics were given asnecessary.

Intra-Coronary Administration of Tile Cross-Linked Alginate Solution

Four days following MI an over-the-wire balloon catheter oversized by0.5 mm was advanced into the LAD coronary artery to the location ofprior occlusion. The cross-linked alginate solution (2 ml) was infuseddistally to the occluding balloon, through the central port of theballoon catheter, during intermittent coronary occlusions, each lasting2.5-4 min. The coronary artery was re-perfused for about 3 min betweenocclusions. Upon completion of intra-coronary administration, coronaryangiography was repeated to ascertain vessel potency and unimpeded flowof contrast material. Serum troponin I levels were measured prior to and24 hr following intracoronary administration.

Echocardiography

Echocardiogram analyses were performed immediately following MI prior toinjection treatment, as well as on day 10, 30 and 60 post MI. Theanalyses were performed using a phased-array transducer (2.5 MHz)equipped with an ultrasound system (Sonos 5500, Hewlett-Packard,Andover, Mass.). Images were recorded on VHS videotape. End-diastolicand end-systolic frames were selected from standard apical andparasternal views.

Global LV ejection fractions (LVEF) were assessed visually. LV volumeswere measured by manually tracing the left ventricular cavity using thesingle plane modified Simpson's algorithm when >80% of the endocardialborder could be detected in both the apical 4- and 2-chamber views, andby a single plane when 80% of the endocardial border could be detectedonly in the apical 4-chamber view.

Regional wall motion and regional LV function of each of the 16 leftventricular segments were assessed using score values (1=normal, 2=.hypokinetic, 3=akinetic, 4=dyskinetic) according to Schiller et al. (J.Am. Soc. Echocardiogr. 2:358-67 1989). Left ventricular wall motionscore index (WMSI) values were calculated using the sum of theindividual scores divided by the• total number of analyzed segments.Regional motion score index were calculated by the same method for thesegments of the mid-LAD territory (infarct related artery territory).

In the four-chamber view, pulsed Doppler• transmittal flow velocitieswere recorded by positioning the sample volume both at the level of thetip of the mitral valve leaflets and the mitral valve annulus. At thelevel of the tip of the mitral valve, time periods were measured betweenthe peak of the ECG R-wave to the beginning, peak and end of thecomponents of the transmittal flow velocity pattern, as well as the timeperiods to the E-wave (early diastolic filling) and A-wave (latediastolic filling). The acceleration and deceleration time periods ofthe E-wave were also measured. The deceleration time were calculated byextrapolating the deceleration slope to the baseline. At the level ofthe mitral valve annulus, diastolic filling time was determined as theduration of mitral flow. The flow during the E-wave and A-wave wascalculated by planimetry of each velocity curve. Each value wasdetermined as the mean measurement of three consecutive steady-statebeats. The occurrence of mitral regurgitation during ischemia wasmonitored using two-D color flow. The data were interpreted by a singleexperienced observer (MSF), and all measurements were obtained offlineby a single technician.

SPECT Imaging

SPECT imaging was performed using conventional techniques usingtechnetium-99m sestamibi. Standard SPECT myocardial scintigrams.

Imaging Protocol:

One hour after intravenous administration of ^(99m)Tc-sestamibi (25 to30 mCi), electrocardiogram-gated single-photon emission computedtomography imaging were performed. All animals were imaged in the supineposition. The radionuclide images were acquired based on our clinicalprotocol with 64 angular views over 180° (from 45° right anterioroblique to 135° left posterior oblique) using a 128×128 matrix, 20 s perview, a 20% energy window centered at 140 keV and a low-energy,high-resolution (LEHR) collimator. Using a two-head gamma camera, thetotal data acquisition time were approximately 12 min.

Semi-Quantitative Analysis:

Images were evaluated in a blinded fashion by two nuclear cardiologistsusing a standard 20-segment analysis (18 short-axis and 2 long-axissegments). Each segment was scored on a 5-point scale of 0 to 4(0=normal perfusion, 1=mild hypoperfusion, 2=moderate hypoperfusion,3=severe hypoperfusion and 4=no perfusion). A sum rest score wascalculated for each animal. A semi-quantitative change score wascalculated as the mean difference between the rest score from the day 90scan and the rest score of the same animal on day 0. LV regional wallmotion was evaluated from the gated study using a similar standard20-segment analysis (18 short-axis and 2 long-axis segments). Eachsegment were scored on a 5-point scale of −1 to 3 (−1=dyskinesis,0=akinesis, 1=moderate/severe hypokinesis, 2=mild hypokinesis and3=normokinesis).

Quantitative Analysis:

Quantitative computer analysis was performed using QPS software (CedarsSinai, LA, Calif.). The software consists of an automatic program,capable of batch processing, which, among various calculations oncardiac single-photon emission computerized tomography perfusion images,performs automatic scoring of these images. The algorithm is independentof myocardial shape, size, and orientation, and establishes a standardthree-dimensional point-to-point correspondence among all sampledmyocardial segments. Percent change in summed rest scores werecalculated as: {[(mean score day 90)−(mean score of day 0)]/(mean scoreof day 0)}*100.

MRI

ECG-gated cardiac MRI was performed using a Signa MR/i, 1.5T EchoSpeedPlus MRI scanner (General Electric Medical Systems, Waukesha, Wis.).Cine MRI images were acquired, using a steady-state cine-MRI technique(Fast Imaging Employing Steady-state Acquisition, FIESTA) acquired inshort-axis oblique (SAO), vertical long-axis-oblique (LAO) and axial4-chamber view. This was followed by perfusion and delayed enhancementanalyses. Perfusion analyses were performed during intravenous injection(10 ml) of gadopentate dimeglumine (Omniscan, 0.5 mmol/ml, NycomedImaging AS, Oslo, Norway) at a rate of 4 ml/sec, using an automaticinjector (Medrad Spectris, Indianola, Pa., USA). Delayed enhancementimages were obtained following an additional injection of gadopentatedimeglumine (0.2 mmol/kg up to a maximum of 20 ml) using inversionrecovery prepared breathhold cine gradient-echo images. LAO and SAOviews were obtained 15 and 20 min following gadolinium injection,respectively.

Image Analysis:

The left and right ventricular end diastolic volume and the left/rightventricular ejection fraction were calculated from cine-MRI images by anexperienced radiologist blinded to other imaging findings. Thecalculations were performed using an Advantage Windows Workstation (Rev4.1, GE, Buc France) and Mass Analysis software (MEDiS, Netherlands).

The Extent of myocardial infarction was estimated using the standard 16segments (Schiller et al., J. Am. Soc. Echocardiogr. 2:358-67 1989) anda three-scale scoring index (0=no evidence of infarction,1=subendocardial infarction, 2=transmural infarction).

Morphological and Histological Examination

Animal hearts were arrested by potassium chloride and rapidly excised.Coronary arteries were perfused with 100 mL 10% formaldehyde and thehearts were the fixed in diastole with an intraventricular pressure of30 mm Hg in formaldehyde solution. Following fixation, the heart tissuewas dissected (5 μm thick), embedded in paraffin blocks at 56° C. andstained with hematoxylin and eosin for histological examination.

The presence and distribution of biotinylated alginate within myocardialtissue was detected using enzyme (peroxidase)-linked avidin.Accordingly, tissue samples were serially rehydrated in 100%, 95%, and70% ethanol following by deparaffinization with toluene. Endogenousperoxidase was then blocked (using methanol, H₂0₂ and H₂0) and thetissue was exposed to peroxidase-linked avidin complex (VectorLaboratories) then counterstained with hematoxylin. Positivebiotinavidin interaction resulted in brown color which highlighted thebiotinylated alginate material.

Results:

Nine pigs were used overall, out of which one animal (pig No. 6) diedduring anesthesia prior to MI. Another animal (pig No. 9), which wassubjected to 90 min occlusion, developed an extensive heart failure anddied five hr following injection (see Table 6 hereinbelow).

Echocardiography, SPECT imaging and MRI analyses did not detect anysignificant cardiac malfunction, arrhythmias, ventricular tachycardia,fibrillation or premature ventricular contractions, in any of thetreated animals during the observation period (between intra coronaryadministration and harvest; up to 5 days).

TABLE 6 Summary of treatments and function analyses Occlusion Intracoronary Pig Time administered Observation No. (Min) material Echo SPECTMRI period³ Comments 1 60 Alginate-Biotin¹ + 1 hr 2 60 Alginate² 5 days3 60 saline + 5 days 4 60 Alginate + 1 hr 5 60 Alginate + 1 hr 6 n/a n/an/a Animal died prior to MI 7 60 Alginate-Biotin + + + 1 hr 8 90Alginate-Biotin + 1 hr 9 90 Alginate + 1 day Animal died 4 hr followingIC administration ¹Alginate-Bioton = biotin-labeled cross-linkedalginate solution. ²Alginate = cross-linked alginate solution only. ³Thetime period between IC administration to harvest.

Morphological and Histological Examinations

Upon visual inspection, transient occlusion of the mid LAD coronaryartery caused significant infarction (ca. 25% of the left ventricle with60 min occlusion) at the anterior, apical, septal and right ventricularapex.

Morphological examinations revealed abundance of gelatinous cross-linkedalginate (biotinylated) present within ischemic myocardial tissue (seenas pale white substance). The morphological observations correlated withSPEC and MRI imaging analyses.

Histological examinations revealed brown stained tissue indicatingextensive spread of the biotinylated cross-linked alginate within theextracellular matrix of the infarcted myocardium tissue. In addition,the cross-linked alginate material was observed in association withdividing cardiomyocytes (noticeable by having multiple nuclei)indicating that the cross-linked alginate solution may promotemyocardial tissue regeneration (FIG. 35).

These results clearly show that the cross-linked alginate solution canbe administered into a blood vessel via catheterization and capable offlow within blood vessels, cross blood capillary walls, spreadthroughout the extracellular matrix of target tissue and assume a gelstate following deposition within the tissue.

Hence, the results indicate that the cross-linked alginate solution canbe delivered into a damaged myocardial tissue via intra coronaryadministration effectively, conveniently and safely.

Example 7 Injection of Cross-Linked Alginate Solution into MouseIschemic Hind Limb Materials and Methods: Aqueous Cross-Linked AlginateSolution

The cross-linked alginate solution was made of 1% w/v sodium alginate(Avg. Mw=30 kDa; G/M ratio 2.1) and 0.3% w/v calcium gluconate, asdescribed in Example 1 hereinabove.

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Mouse Hind Limb Model

A mouse hind limb ischemia model was established essentially asdescribed by Couffinhal et al. (American Journal of Pathology 152:1667-1679, 1998) and Babiaket et al. (Cardiovasc Res. 61:789-795, 2004);Twelve wk old Balb/C mice weighting between 26 and 30 g were used. Undershort-term anesthesia, the left femoral artery were exposed, dissectedfree, and excise according to Madeddu et al. (Faseb. J. 18: 137-139,2004). The experimental setting was intended •to mimic the clinicalsituation of patients with extensive, acute arterial occlusion.

Administration of Cross-Linked Alginate Solution

Cross-linked alginate solution (0.05 ml) was injected intramuscularly tothe ischemic hind legs of treated mice one day following femoral arteryexcision. Untreated (control) mice were injected with PBS.

In Vivo Perfusion Analysis

Perfusion analysis was performed following femoral artery ligation,prior to and 7 days following injection. Quantitative perfusion imagingwere performed via i.v. injection ofHexakis-(2-methoxyisobutylisonitrile)-technetium-99m (Tc-99m-MIBI;Cardiolite®, Dupont Pharma) and a lipophilic perfusion marker (bolus of200 I containing a dose of 50-60 MBq). Data quantification was performedusing a gamma camera (Basicam®, Siemens, using the ICON softwarepackage).

Statistical Analysis

All values are presented as mean ? SE. Changes in values measured in thesame animal during different periods were analyzed by paired t testusing GraphPad. Prism version 4.00 for Windows (GraphPad Software, SanDiego Calif. USA). Differences between the cross-linked alginateinjected (treated) and the PBS injected (control) groups were analyzedby unpaired t test. All tests were two-tailed and p<0.05 was consideredstatistically significant.

Results:

Administration of cross-linked alginate solution into mouse ischemichind limbs significantly increased blood perfusion in the treated limbs,as compared with the control (FIGS. 53-54; p<0.01). In addition, thecross-linked alginate solution effectively protected the ischemic limbsfrom autoamputation, as compared with the control where 3 out of 8 theischemic limbs developed necrosis and were subsequently lost (FIG. 55).

Hence, the results clearly indicate that cross-linked alginate solutioncan be used to repair ischemic muscle tissues.

Example 8 Injection of Cross-Linked Alginate Solution into a RatChronically Damaged Myocardium Materials and Methods: AqueousCross-Linked Alginate Solution

The cross-linked alginate solution was made of 1% w/v sodium alginate(Avg. Mw=30 kDa; G/M ratio 2.1) and 0.3% w/v calcium gluconate, asdescribed in Example 1 hereinabove.

Animal Care

The study was performed in accordance with the guidelines of The AnimalCare and Use Committee of Ben-Gurion University and Sheba MedicalCenter, Tel Aviv University, which conforms to the policies of theAmerican Heart Association and the “Guide for the Care and Use ofLaboratory Animals” (Department of Health and Human Services, NIHPublication No. 85-23).

Induction of Myocardial Infarction

Forty rats were subjected to myocardial infarction using the procedureas described in Example 2 hereinabove.

Injection of the Cross-Linked Alginate Solution

Two months following MI, 27 animals which survived were randomized andinjected with cross-linked alginate solution (treatment group) or withPBS (control group). For injection, animals were anesthetized and theirchest was opened under sterile conditions. The infarcted area wasidentified based on the appearance of surface scar and wall motionakinesis. The scar tissue was then injected, using a 27-gauge needle,with 100-200 μL of the aqueous cross-linked alginate solution, or PBS(control). Following injection, the surgical incision was suturedclosed.

Echocardiography

Transthoracic echocardiography was performed on all animals within 24hours post MI (baseline echocardiogram) and two months post injectionusing the procedure essentially as described by Etzion et al. (J. Mol.Cell Cardiol. 33: 1321-1330, 2001) and Leor et al. (Circulation102:III56-61, 2000). The measured parameters were: LV anterior wallthickness; maximal LV end-diastolic dimension; minimal left ventricularend-systolic dimension in M-mode and 2-D imaging; and fractionalshortening (as a measure of systolic function) calculated as[FS(%)=(LVIDd−LVIDs)/LVIDd×100], where LVID indicates LV internaldimension, s is systole, and d is diastole. Index of change in LV area(%) was calculated as [(EDA−ESA)/EDA]×100 where EDA indicates LV enddiastolic area, ESA indicates LV end systolic area (Mehta et al., J. Am.Coll. Cardiol. 11:630-636, 1988). All measurements were averaged forthree consecutive cardiac cycles.

Morphological and Histological Examination

Two months following injection (four months following MI), animals weresacrificed with an overdose of phenobarbital. Hearts were harvested,sectioned into 5 μm slices and stained with hematoxylin and eosin forhistological examination.

Angiogenesis Assessment

Angiogenesis was assessed by immunohistologic •staining of tissuesections with anti α-actin smooth antibody (Sigma) using the proceduredescribed by Leor et al. (Circulation; 94:II332-336, 1996). Followingpreliminary microscopic examination under low power, five consecutiveadjacent fields were photographed from each section at a magnificationof ×200. The density of blood vessels was estimated using computerizedimage analysis.

Statistical Analysis

All values are presented as mean SE. Changes in values measured in thesame animal during different periods were analyzed by paired t testusing GraphPad Prism version 4.00 for Windows (GraphPad Software, SanDiego Calif. USA). Differences between the cross-linked alginateinjected (treated) and the PBS injected (control) groups were analyzedby unpaired t test. All tests were two-tailed and p<0.05 was consideredstatistically significant.

Results

Administration of cross-linked alginate solution into a damagedmyocardium (two months post MI) significantly increased the density ofblood vessels in the myocardium scar tissue [106±5 vessels/mm² intreated animals, as compared with only 43±8 vessels/mm² in PBS-treated(control) animals; p<0.0001; FIG. 37].

In addition, the cross-linked alginate solution significantly increasedthe average scar thickness (from 16.3±3.1 mm in the control animals to27.9±2.7 mm in the treated animals; p<0.01).

2D echocardiography analyses show that the average LV fractionalshortening in control animals declined (relative to pre MI levels) by37±9%, while the average decline in treated animals was only 12±6%(p<0.05; FIG. 38).

Analyses of E/A wave ratio in Doppler echocardiogram show that theaverage cardiac diastolic function in control animals declined (relativeto pre MI levels) by 3.34±0.5%, while the average decline in treatedanimals was only 1.73±0.09% (p<0.0001; FIG. 39).

The results clearly indicate that the cross-linked alginate solution iscapable of promoting angiogenesis, increasing scar thickness andpreserving the systolic and diastolic function in a chronically damagedmyocardium.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications; patents, patent applicationsmentioned in this specification are herein incorporated in theirentirety by reference into the specification, to the same extent as ifeach individual publication, patent, patent application was specificallyand individually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention.

What is claimed is:
 1. A composition comprising an aqueous solution ofan alginate cross-linked by multivalent cations with an elastic responseequal to or greater than a viscous response, wherein the alginate isprovided by an alginate salt and is present in an amount ranging from0.1 to 4% (w/v) with a molecular weight ranging from 1 to 300 kDa, themultivalent cations are provided by a multivalent cation salt, and amultivalent cation salt to alginate salt ratio ranges between 2:1 and1:10.
 2. The composition of claim 1, wherein the multivalent cations areselected from the group consisting of calcium, strontium, barium,magnesium, aluminum, difunctional organic cations, trifunctional organiccations, and tetrafunctional organic cations.
 3. The composition ofclaim 1, wherein the alginate has a molecular weight ranging between 10and 100 kDa.
 4. The composition of claim 1, wherein the alginate ispresent in an amount ranging from 0.5 to 2% (w/v).
 5. The composition ofclaim 4, wherein the alginate is present in an amount ranging from 0.8to 1.5% (w/v).
 6. The composition of claim 1, wherein the multivalentcation salt to polymer salt ratio ranges between 1:2 and 1:5.
 7. Thecomposition of claim 6, wherein the multivalent cation salt to polymersalt ratio ranges between 1:3 and 1:4.
 8. The composition of claim 1,wherein the alginate has a molecular weight ranging from 10 to 100 kDa.9. The composition of claim 1, wherein the aqueous solution of alginatecross-linked by multivalent cations exhibits an elastic response equalto or greater than its viscous response under small deformationoscillatory frequencies in the linear viscoelastic limit and shearthinning behavior in a power-law relationship.
 10. The composition ofclaim 9, wherein the small deformation oscillatory frequencies rangefrom 0.1 to 10 Hz.
 11. The composition of claim 1, wherein the aqueoussolution maintains a liquid state in storage at a temperature rangingfrom about 4 to about 8° C. for at least 30 days.
 12. The composition ofclaim 1, further comprising at least one therapeutic agent.
 13. Thecomposition of claim 12, wherein the at least one therapeutic agent isselected form the group consisting of a growth factor, a hormone, ananti-inflammatory drug, an anti-apoptotic drug and an antibiotic. 14.The composition of claim 1, further comprising cells.
 15. Thecomposition of claim 14, wherein the cells are selected from the groupconsisting of cardiomyocytes, myoblasts, fibroblasts, chrondrocytes,muscle cells, smooth muscle cells, endothelial cells, mesenchymal cellsand stem cells.
 16. A kit comprising: (a) the composition of claim 1;and (b) a packaging material identifying the kit for administration intoa body tissue.
 17. The kit of claim 16, wherein the tissue is myocardialtissue or muscle tissue.
 18. A method of administering a cross-linkedalginate solution into a body tissue, comprising administering thecomposition of claim 1 into a blood vessel, into muscle tissue, or intomyocardial tissue.
 19. The method of claim 18, wherein administrationcomprises treating congestive heart failure or ischemic mitralregurgitation.
 20. The method of claim 18, wherein the compositioncomprises alginate having a molecular weight ranging from 20 to 50 kDa.21. The method of claim 20, wherein the amount of the alginate is about1% (w/v).
 22. The method of claim 21, wherein the multivalent cationsalt to polymer salt ratio ranges between 1:3 and 1:4.